The overall goal of the proposal is to understand calcium regulation in both cardiac and skeletal muscle at the atomic level. Calcium is an essential messenger for muscle contractility and its homeostatic balance is controlled by proteins embedded in the sarcoplasmic reticulum (SR) membrane. In cardiac myocytes, the SR Ca2+-ATPase 2a isoform (SERCA2a) is responsible for ~70% of the Ca2+ translocation and regulates diastole. SERCA2a is inhibited by phospholamban (PLN), a membrane protein that reverses its inhibition upon phosphorylation at Ser16 and Thr17. In skeletal muscle, the SERCA1a isoform administrates the relaxation phase and is regulated by sarcolipin (SLN), a membrane inhibitor that is post-translationally regulated by phosphorylation at Thr5. Both PLN and SLN maintain SERCA's activity within a physiological window of apparent Ca2+ affinity. When SERCA2a functions outside this window, disruptions in Ca2+ homeostasis leads to dilated or hypertrophic cardiomyopathies, and ultimately heart failure. SERCA1a dysfunctions result in reduced skeletal contractility, leading to conditions such as Brody disease. In past funding cycles, we characterized the structural dynamics of both PLN and SLN in the presence and absence of the ATPase. The latter enabled us to design and test new dominant-negative PLN mutants with promising results towards improving muscle contractility via rAAV-mediated gene therapy. In this competitive renewal, we propose to analyze the effects of the phosphorylation states in both PLN and SLN through investigating the interactions between these two inhibitors and the enzyme along the catalytic cycle, and take advantage of this knowledge in designing mutants with improved loss-of-function characteristics. To carry out these studies, we will utilize a combination of molecular biology, biochemical assay, as well as spectroscopic methods (NMR, EPR, and fluorescence) that will enable the analysis of these membrane protein complexes in native lipids.